Baculovirus insect control compositions with enhanced lethality

ABSTRACT

A method of enhancing baculovirus disease in pests on a crop is provided through an insect control composition by applying an insect control composition to the crop. The applied composition includes a baculovirus that is infectious for at least one pest feeding on the crop and an enhancing agent for the baculovirus. The enhancing agent is preferably administered so as to be ingestible by the pest together with the baculovirus. A preferred enhancing agent is mannitol which increases the mortality of larval tobacco budworms feeding on cotton when infected with Autographa californica by 238%.

FIELD OF THE INVENTION

The present invention generally relates to uses of baculoviruses forinsect control, and more particularly to insect control compositionsincluding baculoviruses with significantly enhanced pest mortality forcommercially practical use on crops such as cotton.

BACKGROUND OF THE INVENTION

The lepidopteran family Noctuidae includes some of the most destructiveagricultural pests, such as the genera Heliothis, Helicoverpa,Spodoptera, and Trichoplusia. For example, included in this family arethe tobacco budworm (Heliothis virescens), the cotton bollworm(Helicoverpa zea), the cotton leafworm (Alabama argillacea), the spottedcutworm (Amathes c-nigrum), the glassy cutworm (Crymodes devastator),the bronzed cutworm (Nephelodes emmedonia), the fall armyworm(Spodoptera frugiperda), the beet armyworm (Spodoptera exigua), and thevariegated cutworm (Peridroma saucia).

Baculoviruses are arthropod-specific, double stranded DNA viruses thatcan be used to control insect pests. The nuclear polyhedrosis viruses("NPV") are one baculovirus subgroup. On the order of forty nuclearpolyhedrosis viruses have been isolated from insect species. (See, forexample, Atlas of Invertebrate Viruses, Adams and Bonami, editors, CRCPress, Inc., 1991.) Various baculoviruses, including those that infectcotton bollworm, Helicoverpa zea, tobacco budworm, Heliothis virescens,Douglas fir tussock moth, Orygia pseudotsugata, gypsy moth, Lymantriadispar, alfalfa looper, Autographa californica, European pine sawfly,Neodiiprion sertifer, and codling moth, Cydia pomonella, have beenregistered as pesticides.

A characteristic feature of the NPV group is that many virions areembedded in a crystalline protein matrix referred to as an "occlusionbody." Examples of NPVs include Lymantria dispar NPV (gypsy moth NPV),Autographa californica MNPV, Anagrapha falcifera NPV (celery looperNPV), Bombyx mori NPV, Spodoptera littoralis NPV, Spodoptera frugiperdaNPV, Heliothis armigera NPV, Mamestra brassicae NPV, Choristoneurafumiferana NPV, Trichoplusia ni NPV, Helicoverpa zea NPV, andRachiplusia ou NPV. For field use, occluded viruses often are preferabledue to their greater stability since the viral occlusion body providesprotection for the enclosed infectious nucleocapsids.

Recombinant baculoviruses are also known and useful as insect controlcompositions. For example, U.S. Pat. No. 5,674,747, issued Oct. 7, 1997,inventors Hammock et al., describes recombinant baculoviruses in which amutated JHE juvenile hormone esterase) coding sequence providesrelatively rapid speed of kill in insects. U.S. Pat. No. 5,674,485,issued Oct. 7, 1997, inventors Hammock et al., describes a recombinantexpression vector capable of expression in a host insect and including acoding sequence for JHE that, when expressed, lacks the signal sequencetargeting the enzyme to the plasma membrane. U.S. Pat. No. 5,643,776,issued Jul. 1, 1997, inventors Hammock et al., describes recombinantbaculoviruses with a mutated JHE coding sequence. McCutchen et al.,Bio/Technology, 9, pp. 848-852 (1991), reports the construction of arecombinant, polyhedron-positive Autographa californica NPV thatexpresses an insect-selective toxin (AaIT), which is isolated from thescorpion Androctonus australis. Copending application Ser. No.08/435,040, filed May 8, 1995, inventors Hammock et al, describes amethod to accelerate the rate of pest kill by treating the pests ortheir loci with at least two different insect toxins, which areexpressed from at least one recombinant microbe such as a baculovirus.

However, a limitation of the use of baculoviruses for pest control hasbeen that their efficacy can be adversely affected by host-plantchemicals and they can also be inactivated by sunlight. The influence ofhost plants on the course and severity of baculovirus disease in insectshas been discussed, for example, by Felton et al. J. Chem. Ecol., 13,pp. 947-957 (1987), J. Chem. Ecol., 16, pp. 1211-1236 (1990), and Arch.Insect Biochem. Physiol, 29, pp. 187-197 (1995). Ignoffo and Garcia,Environmental Entomology, 23, pp. 1025-1029 (1994), have studied theinactivation of microbial insecticides by exposure to sunlight. Theyfound that propyl gallate provided the best protection of Baculovirusheliothis (of three anti-oxidants tested) and catalase the best of threeoxidative enzymes tested. They suggested that reactive radicalsgenerated by sunlight can cause inactivation of field-applied viral andother microbial insecticides. Unfortunately, they acknowledged that thematerials they had used could not be used practically to provide UVprotection of commercial microbial insecticides.

SUMMARY OF THE INVENTION

It is an object of the present invention to substantially enhance themortality of insect pests that ingest baculoviruses during feeding oncrop foliage.

In one aspect of the present invention, a method of enhancingbaculovirus disease in pests on a crop is provided by applying an insectcontrol composition to the crop. The applied composition includes atleast one baculovirus species and a baculovirus disease enhancing agent.The at least one baculovirus species is preferably in the form ofpolyhedral occlusion bodies and can be a mixture of wild-type and/orrecombinant. The enhancing agent is preferably administered so as to beingestible by the pest together with baculovirus as the pest feeds onthe crop, and preferably is in an amount effective to increase pestmortality by at least about 100% with respect to ingestion of thebaculovirus alone.

A particularly preferred embodiment of the invention is wherein mannitolis used as an enhancing agent. When mannitol was applied in combinationwith a baculovirus infectious for tobacco budworm, then larval tobaccobudworms feeding on cotton had mortality increased by 238%.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 graphically illustrates the percent of viral disease inhibitionwhen larvae of tobacco budworm (Heliothis virescens, feeding on cottonor lettuce) was infected with Autographa californica AcMNPV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have discovered that the susceptibility of noctuidae larvae tobaculovirus infection is inhibited in large part by phenolic oxidationby phenolases, particularly peroxidase (POD), which subsequentlygenerates free radicals in the foliage being ingested by the infectedpest. The more free radicals generated, the lower the incidence of viraldisease, particularly when the crop is cotton.

With reference to FIG. 1, one can see that baculovirus insecticides donot perform well for controlling noctuid pests on a crop such as cotton.The viral disease is almost 60% inhibited compared with larvae fed ondiet or iceberg lettuce. The diminished efficacy of baculoviruses on acrop such as cotton is the result of free radicals generated during thesimultaneous ingestion of plant foliage with viral particles by the hostinsect. Free radical generation leading to inhibition of viral diseaseis mediated, at least in part, by POD.

We developed an assay for the detection of free radicals in plantfoliage in the immediate aftermath of an oxidative burst. Assessment offree radical generation, such as in an assay of crushed foliage, is auseful predictor of the inhibition of baculoviruses as pest controlagents on plants. Cotton appears to be particularly inhibitory tobaculovirus disease, perhaps as a consequence of complex redox cyclingin the plant.

The present invention provides combinations of baculoviruses withbaculovirus disease enhancing agents that overcome the inhibitory effectin crop foliage, particularly for a crop such as cotton. Becausebaculoviruses do not at present provide adequate control on crops suchas cotton, and since cotton is a major crop worldwide, the presentinvention is of considerable practical importance to enhance baculovirusinsecticides on a crop such as cotton.

Broadly, the invention comprises administering an insect controlcomposition to the crop for pest control where the composition includesat least one baculovirus species and a baculovirus disease enhancingagent. The baculovirus species in the insect control compositionpreferably is in the form of polyhedral occlusion bodies, since theviral occlusion body provides protection for the enclosed infectiousnucleocapsids during field applications. Of particular interest for acrop such as cotton are the baculoviruses that infect cotton bollworm(Helicoverpa zea) and tobacco budworm (Heliothis virescens).

In practicing the present invention, at least one baculovirus species isused. More than one baculovirus species can be used so as to increasethe insecticidal effects against a wider variety of insects. Suchcombinations of baculoviruses are known to the art. For example, Maedaet al. in European Patent Application Publication No. 0 225 777,published Jun. 16, 1987, discloses a recombinant virus containing DNAsegments of both of two species of viruses for which their host insectsdiffered. U.S. Pat. No. 5,071,748, issued Dec. 10, 1991, inventorMiller, discloses mixtures of baculoviruses. U.S. Pat. No. 5,756,340,issued May 26, 1998, Ser. No. 08/435,040, filed May 8, 1995, inventorsHammock et al., discloses insect control through combinations ofinsecticidal recombinant microbes.

The one or more baculoviruses of the inventive insect controlcomposition may each be wild-type, may be recombinant, or may bemixtures of wild-type and recombinant. Where more than one baculovirusis used, then one usually will choose the combination so as to avoid thetwo infecting the same insect. This is because such usually can lead tointerference between the baculoviruses and consequently decreasedlethality; however, there are instances where there can becomplementarity in mixtures of baculoviruses where both infect the sameinsect.

Among suitable recombinant baculoviruses are those expressing a scorpiontoxin, such as the excitatory toxin from Androctonus australis (AaIT) orfrom Leuirus quinquestriatus hebraeus (LqhαIT), such as are described byHammock et al. in U.S. Pat. No. 5,756,340, issued May 26, 1998, Ser. No.08/435,040, McCutcheon et al., Bio/Technology, 9, pp. 848-852 (1991),and Maeda et al., "Insecticidal Effects of an Insect-Specific NeurotoxinExpressed by a Recombinant Baculovirus," Virology, 184, pp. 777-780(1991). These latter two illustrate construction of a recombinantbaculovirus expressing AaIT.

The greatest inhibition of disease in the field appears to occur atlower viral doses. Growers typically apply a very high viral dose (LD₉₉)initially, but production costs will limit the amount of virus that canbe commercially applied. As will be discussed further, even smallamounts of the enhancing agents of the invention will be effective atsubstantially any viral dose. In other words, enhancing agents of theinvention are effective even at lower viral doses (such as LD₂₅) wherethe greatest inhibition of disease caused by the plant occurs.

The second essential component of the present invention is a baculovirusdisease enhancing agent which is administered either simultaneously withthe baculovirus or baculovirus mixture (such as in an admixture ofbaculovirus occlusion bodies) or is administered within about threehours prior to application of baculovirus to the crop for which pestcontrol is desired.

Many of the preferred enhancing agents for practicing this invention areanti-oxidants; however, although Ignoffo and Garcia, supra, had foundthat propyl gallate (an anti-oxidant) protected the baculovirusheliothis (HzSNPV) against sunlight, we have found substantially noprotection of baculovirus from inhibition by propyl gallate. Ignoffo andGarcia also had found some efficacy with the oxidative enzyme catalase;however, although we found that catalase did provide some enhancementwhen applied with baculovirus to cotton foliage, the enhancement wasonly about 68% compared with untreated cotton.

Although an anti-oxidant (such as propyl gallate) or an oxidative enzyme(such as catalase) may inhibit sunlight inactivation of viral pesticideswhen these compounds are admixed with occlusion bodies and then exposedto fluorescent lamps, such conditions are quite different from thosethat occur in the field when caterpillars ingest polyhedrin occlusionbodies along with the crop foliage. For one example, pest caterpillarstypically have a stomach pH of about 9. Thus it is not surprising thatthe work of Ignoffo and Garcia with simulated sunlight would not bepredictive of results when agents, in combination with baculoviruses,are ingested together with crop foliage. In order to determine whetherenhancing agents are useful for purposes of the present invention, theycan be assessed, without undue experimentation, for free radicalgeneration, such as in the assay using crushed foliage that will be morefully described hereinafter. Thus, one can reasonably predict thathydroxyl scavengers, and other similar free radical scavengers, will beuseful as enhancing agents in practicing the present invention.

Disease enhancing agents of the invention are in an amount substantiallyto increase post mortality, preferably in an amount effective toincrease pest mortality by at least about 100% (with respect to pestmortality from administration of the baculovirus species withoutenhancing agents). Enhancing agents suitable for practice of the presentinvention fall into several functional types (which can overlap infunction).

One type is free radical scavengers, particularly hydroxyl radicalscavengers, which normally will need to be effective in the pest gut.This type includes polyhydroxy compounds. Thus, polyhydroxy compoundssuch as glucose, sucrose, lactose, ribose, galactose, and mannitol areuseful enhancing agents. Particularly preferred is mannitol. Othersuitable hydroxyl radical scavengers can be screened by methods wellknown to the art. For example, Halliwell et al., AnalyticalBiochemistry, 165, pp. 215-219 (1987) describe a simple assay fordetermining reactions of reagents with hydroxyl radical scavengingfunction. Halliwell et al. further describe measurements of hydroxyradicals in biochemical systems, Methods of Biochemical Analysis, 33,pp. 59-90.

Another type of enhancing agent for practicing the invention is achelating agent, for example, those agents which are effective tochelate with phenolic in the pest gut. Among suitable chelating agentsare sodium borate, or metal chelators such as thiourea.

We have also found that some anti-oxidants can provide good inhibitionretardation, including ascorbate, and most notably butylated hydroxyanisole and butylated hydroxy toluene (BHA and BHT, respectively).

The invention will now be illustrated by the following examples, whichare intended to illustrate but not to limit the subject invention.

EXAMPLE 1

The free radicals causing baculoviral disease inhibition include activeoxygen species (AOS). This example includes the description of an assayfor measuring AOS in crushed foliage.

Using a homemade leaf press that crushes a moistened leaf between twosolid metal cylinders turning in unison (designed by T. L. German,University of Wisconsin), the pressed-leaf-extract was collected fromeach pre-weighed leaf sample as it was washed with 2.5 ml of a 0.02%solution of methemoglobin in pH 7 buffer into a 20 ml scintillationvial. The cylinders of the leaf press were washed with distilled waterbetween samples. Immediately after collecting the pressed-leaf-extract,the sample was placed in an Eppendorf™ tube followed by centrifugationat 14,000 rpm for 3-5 seconds to precipitate remaining leaf material.The supernatant was immediately transferred to a 1.5 ml cuvette and theabsorbance read at 406 nm against a buffer blank. Samples were incubatedfor 1 hour in the dark. After 1 hour, the absorbance was read again.Because oxidation of the phenolic compounds from thepressed-leaf-extract occurs during the incubation period by the enzymesreleased by crushing, we also read the initial and final absorbances ofsamples in the absence of heme. The increase in absorbance due tooxidation was subtracted from the decrease in absorbance obtained fromheme.

To determine the amount of AOS/free radicals generated in each sample,the difference between the initial absorbance and the final absorbancewas corrected for leaf weight to obtain the change in absorbance/g ofleaf tissue. This value was entered into the equation for the standardcurve to quantify free radical generation in μmoles free radical/AOSequivalents.

Leaves of cotton and lettuce were divided into equal pieces. Eachchemical treatment was applied to a piece of the same leaf Becausetomato has 5-7 leaflets/leaf, rather than cutting the leaf, one leafletfrom each leaf was used as a control. All leaf pieces and leaflets wereweighed prior to crushing. For each chemical treatment applied tofoliage in bioassays (described below), 25 μl of a test chemicalsolution was first added to the scintillation vial used to collect thepressed-leaf-extract. Samples were also run in the absence of heme topermit correction for oxidation as described above. Free radicalgeneration in crushed cotton, lettuce, and tomato foliage were reportedas the mean±SE of four replicates.

Eggs of tobacco budworm (H. virescens) and corn earworm (H. zea) wereobtained from the USDA/ARS (Stoneville, Miss.). Neonate larvae werereared to third instar on semi-synthetic diet (Southland Products, LakeVillage, Ark.) in 24-well tissue culture plates (Fisher) at 26±1° C. and16L:8D. Within 6 hours after molting to the third instar, larvae weretransferred to empty 24-well tissue culture plates for 24 hours to voidtheir gut.

Autographa californica M nuclear polyhedrosis virus (AcMNPV, C6 clone)(Ayers et al., 1994) and Helicoverpa zea S NPV (HzSNPV, original isolateplaque purified from Elxar™, Sandoz-Wander (Wasco, Calif.)) wereamplified in larvae of H. virescens and H. zea, respectively, extracted,partially purified, and stored until use.

Plants were used at the 4-5 leaf stage. A leaf was randomly removed fromeach of 16 plants. Using a No. 3 cork borer, 0.65 cm² leaf disks werecut from each leaf and placed in petri dishes partially filled with 2.4%agar. Leaf disks were distributed such that each chemical treatmentwould be applied to three disks from each plant. Gelatin/chemicalmixtures were prepared by dissolving Knox™ gelatin in 0.1M potassiumphosphate buffer pH 7 at 50%C at a concentration of 4% for cotton and 6%for lettuce and tomato. After cooling at 37° C., test chemicals wereadded to the gelatin as described in Wolfson and Murdock (1987). Fortreatments involving application of semi-purified POD to foliage, anadditional control included boiling the enzyme for 15 minutes beforemixing with gelatin. On lettuce, semi-purified POD was applied at fourdoses (125, 62.5, and 31.25 μg/ml and 100, 62.5, 31.25, and 15.6 μg/ml,respectively) to evaluate whether inhibition of disease responded in adose-dependent manner. Ten μl of each gelatin/chemical mixture at 35-37°C. were applied to each leaf disk with a pipette and distributed evenlywith a fine paint brush. Each treatment mixture was applied to a groupof 48 leaf disks. After drying at ambient temperature (≈30 minutes), aviral formulation was applied to each leaf disk.

AcMNPV was tested on cotton and lettuce against H. virescens; whereas,HzSNPV was tested on tomato against H. virescens and H. zea. Polyhedralocclusion bodies (OBS) were suspended at a single concentration (30OBS/μl for AcMNPV and 3 OBS/μl for HzSNPV) in milliQ H₂ O forapplication in 1 μl aliquots to each leaf disk. We used a low dosebecause we suspect that lower vial doses are probably more biologicallyrelevant (Duffey et al., 1995). The dose for each replicate wasdetermined by treating a group of insects on small cubes (8 mm³) ofartificial diet. Viral inoculum was allowed to dry at ambienttemperature. Once the viral solution was dry, leaf disks were placed oneper well in 24-well tissue culture plates partially filled with 2.4%agar. A starved larva was then transferred one to a well so that eachlarva received a single leaf disk. After 18 hours, insects that hadconsumed an entire treated leaf disk or artificial diet were transferredindividually to excess diet in 35 ml cups and maintained until death orpupation at 26±1° C. and 16L:8D. A group of control insects was treatedwith 80 μM sodium borate in the absence of virus to ensure that thischemical was not toxic to the larvae at this concentration. Mortalitywas scored at 8-9 days post-infection.

The effect of chemical applications to foliage on subsequent phenolaseactivities was examined using a portion of 8 of the 16 leaves used forbioassay. Final concentrations of test chemicals prepared in gelatin andapplied to leaves were determined by measuring the amount of gelatinthat remained on the leaf disk after handling just prior to being fed tolarvae. A 0.1% solution of rose bengal (Sigma) was mixed with gelatinand applied to 10 leaf disks from each plant species as described above.After drying, leaf disks were placed in 0.1M K phosphate buffer andstirred with gentle heating to wash all the gelatin off the leaf disks.Absorbances were read at 496 nm, which is the peak of the visiblespectrum for rose bengal. The mean amount of gelatin that stuck to theleaves was calculated based on a standard curve. On cotton, tomato, andlettuce, 5.88±0.36, 4.58±0.36, and 4.08±0.13 μl of the 10 μl of thegelatin mixture applied to leaf disks still remaining at the time theywere fed to larvae.

Survival data were analyzed by logistic regression. In addition, wecompared the frequency of dead with alive insects between selectedtreatments using data pooled from the two replicates by Chi-squareanalysis with Bonferroni's correction for the number of pairedcomparisons (Steel and Torrie, 1980). We pooled all data for analyses asthe mortality levels characterized by artificial diet showedconsiderable variation between replicates.

We regressed total percent mortality for each treatment on each plantspecies as a function of mean free radical generation (as μmole freeradical/AOS equivalents) (Steel and Torrie, 1980). Free radicals wereassayed using the same plant cultivars under the same conditions as thebioassay on a different day. We regressed mean free radical generationfor each foliar treatment as a function of man foliar POD activity foreach treatment. This analysis was performed on pooled data.

We tested our ability to reverse the effects of POD activity and/or freeradical generation as inhibitors of viral disease by applying a varietyof antioxidants to foliage. On tomato, all antioxidants were applied incombination with POD because tomato by itself is not inhibitory to thevirus.

All antioxidants tested enhanced viral disease on at least one plantspecies. For example, ascorbate completely inhibited all phenolaseactivity when applied to cotton or tomato foliage. On cotton and tomato,mortality increased 63% (x² =2.0, p=0.1499, n=169) and 432% (11,p=0.0079, n=180), respectively. We could not assess accurately theimpact of ascorbate on free radical generation, however, because freeradicals increase markedly in the presence of Fe²⁺ giving ambiguousresults (Koppenol and Butler, 1985).

Application of mannitol, a OH. radical scavenger, provided the bestprotection of all antioxidants tested on cotton. Mannitol did not affectcotton POD activity, but did inhibit free radical generation and enhancelarval mortality compared to untreated cotton foliage by 238% (Table 1;x² =13, p=0.0003, n=172).

Table 1 summarizes the effects of applying enhancing agents inaccordance with the invention to cotton foliage. The pest larvae was H.virescens, which ingested baculovirus (AcMNPV) in conjunction with acontrol (gelatin only) or in conjunction with an enhancing agent of theinvention.

                  TABLE I                                                         ______________________________________                                        Cotton Foliage (with AcMNPV)                                                                    % Mortality of Pest                                         ______________________________________                                        Control           13                                                          Mannitol (20 μM)                                                                                                            44                           Sodium borate (80 μM)                                                                                                  41                                BHT (20 μM)                                        38                      Lutein (0.4 μM)                                                                                                              25                          ______________________________________                                    

Application of BHT enhanced viral disease on all tested plants, bringingthe percent mortality to the level of that obtained on artificial dietor higher. For example, mortality on cotton increased 186% followingapplication of BHT (x² =13.0, p=0.003, n=179). On tomato, BHT increasedmortality by 263% (x² =7.0, p=0.0081, n=168). BHT also inhibited POD andfree radical activities in all tested plant species.

Lutein, a water soluble analog of β-carotene, did not scavenge freeradicals as effectively as BHT based on the heme assay, but was fairlyeffective at reversing free radical inhibition of viral disease. Luteinmoderately inhibited free radical generation in cotton (49%), but wasonly mildly inhibitory in tomato (15%) and lettuce (32%). As aconsequence of lutein application, viral disease was enhanced on cottonby 91% relative to the control such that mortality approached thatobtained on artificial diet (25% compared with 30% on diet). Addition oflutein plus cotton POD to tomato allowed mortality to nearly reach thelevel on untreated control foliage (mortality for POD added to tomatowithout lutein was 6.25%).

Borate, which forms a chelate with catecholic phenolics, was tested oncotton and lettuce foliage. Application of borate, however, markedlyenhanced viral disease on cotton by 215% (x² =11, p=0.0008, n=178); freeradical generation was not affected based on the heme assay. On lettuceborate inhibited POD activity, yet free radical generation increasedaccording to the heme assay. None of the insects died that were treatedwith the same concentration of borate applied to lettuce in the absenceof virus.

EXAMPLE 2

An experiment analogous to that as described in Example 1 was repeated,but instead of the AcMNPV baculovirus for testing on cotton and lettuceor HzSNPV for testing on tomato, a recombinant baculovirus was utilized(AcAaIT).

A group of antioxidants were applied to cotton using the recombinantAcAaIT. The same results were seen as with the wild-type viruses.Third-instar larvae of Heliothis virescens were treated with 30occlusion bodies/larva of AcAaIT (the recombinant expressing aneurotoxin from a scorpion) where the treatments to the leaf were (1)gelatin (control), (2) application of a free radical generator (1%quebracho tannin), or (3) application of 100 μM ascorbate (a reducingagent and free radical scavenger which works sometimes, but not always).Percentage mortality of 50 larvae in each group were as follows:

Control: 10%

Application of Tannins: 0%

Application of Ascorbate: 30.4% (a 3-fold improvement in performance ofthe virus with the addition of an antioxidant).

It is to be understood that while the invention has been described abovein conjunction with preferred specific embodiments, the description andexamples are intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

It is claimed:
 1. A method of enhancing baculovirus disease in pests ona crop, comprising:administering an insect control composition to thecrop for pest control thereof, the composition including (1) at leastone baculovirus species and (2) a baculovirus disease enhancing agent,the enhancing agent being administered before or with the baculovirusspecies and being in an amount effective to increase pest mortality withrespect to pest mortality from administration of the baculovirus specieswithout the presence of enhancing agent.
 2. The method as in claim 1wherein the enhancing agent increases pest mortality by at least about100%.
 3. The method as in claim 2 wherein the enhancing agent is a freeradical scavenger.
 4. The method as in claim 3 wherein the enhancingagent is administered so as to be ingestible by the pest, and theenhancing agent is effective to scavenge free radicals in the pest gut.5. The method as in claim 3 wherein the crop is cotton.
 6. The method asin claim 5 wherein the enhancing agent is a polyhydroxy compound.
 7. Themethod as in claim 2 wherein the enhancing agent includes mannitol. 8.The method as in claim 5 wherein the enhancing agent is administered soas to be ingestible by the pest, and the enhancing agent is effective tochelate with phenolics in the pest gut.
 9. The method as in claim 2wherein the enhancing agent includes sodium borate.
 10. The method as inclaim 2 wherein the enhancing agent includes butylated hydroxyanisole,butylated hydroxy toluene, or mixtures thereof.
 11. The method as inclaim 2 wherein the enhancing agent is a hydroxyl radical scavenger andis in a concentration in the administered insect control composition offrom about 100 μM to about 100 mM.
 12. A method for enhancingbaculovirus disease in pests feeding on a crop, comprising:administeringan insect control composition to the crop for pest control thereof, thecomposition including (1) at least one baculovirus species and (2) abaculovirus disease enhancing agent, the enhancing agent beingingestible by the pest in conjunction with the baculovirus species andbeing in an amount effective to increase pest mortality by at leastabout 100% with respect to ingestion of baculovirus species alone. 13.The method as in claim 12 wherein the enhancing agent is effective as afree radical scavenger in the pest gut.
 14. The method as in claim 12wherein the at least one baculovirus species and the baculovirus diseaseenhancing agent are in an admixture, and the administering includesapplying the admixture to the crop foliage.
 15. The method as in claim12 wherein the crop is cotton.
 16. The method as in claim 13 wherein theenhancing agent is a polyhydroxy compound.
 17. The method as in claim 15wherein the enhancing agent includes mannitol.
 18. The method as inclaim 15 wherein the enhancing agent includes sodium borate.
 19. Themethod as in claim 15 wherein the enhancing agent includes butylatedhydroxyanisole, butylated hydroxy toluene, or mixtures thereof.
 20. Themethod as in claim 12 wherein the enhancing agent is a hydroxyl radicalscavenger, and the crop is cotton.
 21. A method for enhancingbaculovirus disease of noctuid larvae on cotton comprising:administeringan insect control composition to cotton foliage, the insect controlcomposition including occlusion bodies of at least one baculovirusspecies infectious for noctuid larvae, and a disease enhancing agent forsaid baculovirus species when the insect pest is infected therewith, theocclusion bodies being administered to the foliage (a) simultaneouslywith the enhancing agent, or (b) within about three hours subsequent tothe enhancing agent.
 22. The method as in claim 21 wherein the at leastone baculovirus species is effective in infecting Heliothis virescens orHelicoverpa zea larvae.
 23. The method as in claim 21 wherein theenhancing agent is effective in inhibiting peroxidase or peroxidasereaction products in the pest gut.
 24. The method as in claim 23 whereinthe enhancing agent is an hydroxyl radical scavenger.
 25. The method asin claim 21 wherein the enhancing agent includes mannitol.
 26. Themethod as in claim 23 wherein the enhancing agent forms chelates withphenolics.
 27. The method as in claim 21 wherein the insect controlcomposition is administered in a viral dose of at least about the LD₂₅for the noctuid larvae.
 28. The method as in claim 21 wherein the atleast one baculovirus species expresses the disease enhancing agent as aforeign gene product.
 29. The method as in claim 28 wherein theexpression of the foreign gene is of a peroxidase inhibitor, a freeradical scavenger, or a quinone trapping agent, or a reducing agent. 30.A method of controlling pests on cotton, comprising:administering aninsect control composition to cotton foliage, the insect controlcomposition including occlusion bodies of at least one baculovirusspecies infectious for a cotton pest, and a disease enhancing for thebaculovirus when infecting the cotton pest, the disease enhancing agentincluding mannitol, sodium borate, BHT, BHA, ascorbate, or mixturesthereof in an amount from about 100 μM to about 500 mM.
 31. The methodas in claim 30 wherein the baculovirus species is one or more ofAnagrapha falcifera, Anticarsia gemmatalis, Autographa californica,Buzura suppressuria, Cydia pomonella, Helicoverpa zea, Heliothisarmigera, Manestia brassicae, Plutella xylostella, Spodoptera exigua,Spodoptera littoralis, Spodoptera litura, and recombinants thereof.